The human lens must maintain transparency over many decades and, to do so without a blood supply, the lens maintains a fluid microcirculation system to deliver nutrients throughout the tissue. Recently, MRI imaging of water movement has revealed a barrier to water movement in the lens inner cortex that we hypothesize is an important feature of the microcirculation system. However, the molecular details of how this microcirculation system is established and maintained with age are not completely understood. Lens membrane proteins, such as transporters, channels and adhesion molecules, likely play essential roles in the development and maintenance of lens transparency via the microcirculation system. We hypothesize that lens aquaporins (AQPs) play important roles in generating the lens microcirculation system by generating regional differences in water permeability and by establishing an extracellular diffusion barrier. Aquaporin-0 (AQP0) is the most abundant lens membrane protein with reported roles in lens fiber cell adhesion, in water permeability, and in fiber cell organization and, as such, is vital for the development and maintenance of lens transparency. A second aquaporin, AQP5, is also present in lens fiber cells; however, its role in fiber cell function is unclear. The long-term goal of our research is to understand how lens protein modifications that occur during development, aging, and cataractogenesis lead to lens transparency or opacification. In the context of the microcirculation system, we hypothesize that posttranslational modifications and AQP-lipid interactions are important molecular mechanisms used to modulate AQP functionality in each lens region. In addition, we hypothesize that AQP5 trafficking is carefully controlled by posttranslational modification in response to mechanical or osmotic stress. To test our hypotheses we will employ water permeability and cell adhesion assays, native mass spectrometry and proteomics analyses, as well as multi-modal imaging methods to obtain a molecular level understanding of how the structures and functions of lens AQPs change in specific lens regions. We propose three region-specific aims: 1) in the outer cortex of human lenses, use mass spectrometry methods and functional assays to determine AQP structure, function, and regulation; 2) in the extracellular barrier region, use magnetic resonance imaging of lens water movement, super resolution microscopy to map extracellular space, imaging mass spectrometry to map changes to AQPs and lens lipids and functional assays top assess AQP function; and 3) in the core of human lenses, identify AQP modifications and determine the functional roles of modified AQP0 and AQP5. We expect to provide a new molecular level understanding of the lens microcirculation system and the roles of AQPs and their modifications in maintaining lens transparency.